Fine Structure of the Gas Bladder of Alligator Gar, Atractosteus Spatula Ahmad Omar-Ali1, Wes Baumgartner2, Peter J

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International Journal of Scientific Research in Environmental Science and Toxicology Research Article Open Access Fine Structure of the Gas Bladder of Alligator Gar, Atractosteus spatula Ahmad Omar-Ali1, Wes Baumgartner2, Peter J. Allen3, Lora Petrie-Hanson1* 1Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, USA 2Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, USA 3Department of Wildlife, Fisheries and Aquaculture, College of Forest Resources, Mississippi State University, Mississippi State, MS 39762, USA

Received: 1 November, 2016; Accepted: 2 December, 2016 ; Published: 12 December, 2016

*Corresponding author: Lora Petrie-Hanson, Associate Professor, College of Veterinary Medicine, 240 Wise Center Drive PO Box 6100, Mississippi State, MS 39762, USA, Tel: +1-(601)-325-1291; Fax: +1-(662)325-1031; E-mail: [email protected]

Lepisosteidae includes the genera Atractosteus and Lepisosteus. Abstract Atractosteus includes A. spatula (alligator gar), A. tristoechus Anthropogenic factors seriously affect water quality and (Cuban gar), and A. tropicus (tropical gar), while Lepisosteus includes L. oculatus (spotted gar), L. osseus (long nose gar), L. in the Mississippi River and the coastal estuaries. Alligator gar platostomus (short nose gar), and L. platyrhincus (Florida gar) (adversely affect fish ) populations. inhabits these Agricultural waters and run-off is impacted accumulates by Atractosteus spatula [2,6,7]. Atractosteus are distinguishable from Lepisosteus by agricultural pollution, petrochemical contaminants and oil spills. shorter, more numerous gill rakers and a more prominent accessory organ. The gas bladder, or Air Breathing Organ (ABO) of second row of teeth in the upper jaw [2]. Atractosteus spatula alligatorThese fish gar are (Atractosteus bimodal air spatulabreathers,), is and a vascularized use a primitive air sac lung located as an are distributed in lakes, rivers and estuaries along the coast of dorsal to the body lumen. It has characteristics of amphibian lungs. the northern Gulf of Mexico [3,8,9]. Currently, estimated gar

respiratory exchange with aerial gas exchange. The alligator gar gas alligator gar numbers have decreased [5]. bladderAlligator is gar an air-breathingelongated air organssac that function originates to dorsalsupplement to the branchial pharynx. numbers and fishing regulations vary by state. In Louisiana,

their aquatic habitats range from desert to tropical rain forest. Air enters through a pharyngeal-esophageal duct that is controlled by 5mm long and is lined by ciliated columnar epithelium that is AmphibiansAir breathing evolved fishes to breathe occur in air fresh while and inhabiting salt water aquatic [1] and or continuoustwo glottal ridges.with the The central pharyngeo-esophageal canal epithelium. duct The isgas approximately bladder has a central canal that subdivides the bladder into right and left lobes. Each lobe is further divided by septa into series of air spaces. The evolved to breathe by exchanging gases from the water and the terrestrial environments. In contrast, bimodal air breathing fish septa consist of blood vessels and smooth and striated muscles. Air circulates throughout the central canal, lobes and air spaces. The thickness of the septa is determined by underlying, supportive atmosphere [1], while inhabiting water. Obligatory air breathing striated muscle. The internal surface of the gas bladder is a continuous obligatoryfishes use onlyair breathers aerial gas can exchange utilize branchial and bimodal and Air air Breathing breathing respiratory epithelial layer that includes mucus cells, pneumocytes, fishes use primarily aerial gas exchange [10-13]. Bimodal and and ciliated epithelial cells. An understanding of the normal tissue characteristics of this air breathing organ provides a baseline for Organ (ABO) respiration simultaneously [1]. Gars are bimodal air studying the effects of environmental toxins on this organ. breathing fish that use their gas bladder as a respiratory organ Keywords: Alligator gar; Atractosteus spatula; Air breathing into extractdirect contactoxygen fromwith thethe atmosphereenvironment. [1], They especially have inimportant hypoxic functionsenvironments including [14]. Fishrespiration, gills have ion a large regulation, surface areaexcretion and areof organ, Gas bladder; Pharyngeo-esophageal duct Introduction nitrogenous waste, and gas exchange [15-18]. Gill surface areas Alligator gar, Atractosteus spatula, is member of the and lamellar thickness are reduced in air breathing fishes in comparison to non-air breathing fishes, but still function for is phylogenetically placed between the chondrosteans and both O2 uptake and most of CO2 excretion [19-22]. Infraclass Holostei. This group includes primitive fish and amphibianThe gas lungs bladder function of airsimilarly, breathing these fish two hasorgans histological develop the teleosts [1]. Holostei arose around 180 million years ago ontogeneticallyfeatures of amphibian from lungsdifferent [23,24]. tissues Although [25]. theEmbryologically, fish ABO and [2,3], and includes the orders Amiiformes, the bowfin, and TheLepisosteiformes, alligator gar theis the gars. largest These fishof the have gars not (nearlybeen well 3 studiedmeters as a dorsal diverticulum of the esophagus and is joined to the maximally)[4]. Gars occur [5]. in Within North andthe orderCentral Lepisosteiformes, America, as well asthe in family Cuba. esophagusthe gas bladder by the originates pneumatic from duct the pharyngeo-esophageal[26,27]. This duct remains area

were hydroxide, rinsed again in deionized water, dehydrated through surroundingin fish with a the physostomous presence or air absence bladder, of buta pneumatic is absent induct adult in graded then alcohol, rinsed stained in deionized with hematoxylin water, dipped and in eosin, 0.3% andammonia cover fish with a physoclistic air bladder [28]. There is controversy numerous septa that provide a large vascularized area for gas lepisosteid fish (reviewed in [29]. The bladder itself contains Pictureslipped. Frame™ Slides software. were viewed under light microscopy on an Olympus BX 51 (Olympus America Inc.) and photographed using muscle throughout the gas bladder. It is attached to the body Transmission Electron Microscopy (TEM) wallexchange by dorsal [30]. retractors, There is allowing also abundant for contraction. smooth and skeletal Most studies on the physiology of gars have utilized Lepisosteus Samples were primarily fixed in 2% glutaraldehyde in the bladder in oxygen uptake and carbon dioxide release and cacodylate buffer (0.1 M phosphate buffer, pH 7.2) and then post species [1,30]. Such studies confirm the function of fixed in 2% osmium tetroxide at 4°C. Samples were dehydrated functions and presumably physiology are seen in Atractosteus in solutions of increasing ethanol concentration from 35% spatulaits necessity when fish are in hypoxic waters [1,14,22]. Similar to 100% ETOH, and then in solutions of increasing acetone concentration. Finally, samples were embedded in Spurr’s resin. Alligator[1,31]. gar is exposed to petrochemical pollutants and Ultra-sections were made from each block using a Reichert Jung agricultural runoff because of the environments they inhabit. sectionsultra-microtome [35,36]. andAll sectionsstained withwere toluidine viewed underblue stain transmission for thick Furthermore, as top predators they can bio accumulate sections or uranyl acetate and lead citrate for the ultra-thin pollutants. Alligator gar were evaluated in our studies following Resultselectron microscopy on a JEOL JEM-1230 at 80 kV. structure of the alligator gar air breathing organ has not been Gross Morphology documented.the Gulf of Mexico The purpose oil spill [32,33].Toof this study our was knowledge, to histologically the fine describe the alligator gar ABO using light and electron The alligator gar gas bladder is a large, dorsoventrally microscopy. Knowledge of this organ is necessary to evaluate the effects of pollutants on alligator gar. The ABO is the focus of musculoskeletal tissues of the dorsal coelom along its entire flattened, elongate organ that is intimately associated with the

Materialsour on-going and studies Methods of this unusual fish. length and width (Figure1 A, G). The bladder originates at the abrupt termination of the pharyngeo-esophageal duct (Figure Animals collagenous folds that run along its entire length on either side of1D, the asterisks) vertebral and column, is attached blending to the into body the wall stroma by paired that slenderinvests the aorta and cardinal veins associated with the mesonephros

Nine alligator gar were obtained from the Private John Allen width of the body, tapering and terminating at the end of the National Fish Hatchery in Tupelo, MS, and held in fresh water (Figure 1G). The width of the gas bladder is proportional to the at the Mississippi Agriculture and Forestry Experiment Station, South Farm Aquaculture Unit, following published methods coelom (Figure 1 A, B). [34].When sampled, the fish weighed 318 to 320 gm and pharynx between the epi pharyngeal teeth, extending caudally to the Thejunction pharyngeo-esophageal where pharyngeal openingmucosa blendsarises frominto esophagealthe caudal methanesulfonate).measured 425 to 430 The mm gills, in bodylength. wall For and tissue other collection, viscera wereeach fish was placed in an overdose of anesthetic (500 mg/L tricaine mucosa. It begins anteriorly as a single longitudinally oriented,

removed for analysis of the pharyngeo-esophageal junction. extends into the proximal esophagus (Figure 2A). It is delimited slit-like opening between the pharyngeal dental pads and The MSU Institutional Animal Care and Use Committee (IACUC) Light microscopy approved fish holding and experimental protocols. by symmetrical fibrous lateral glottal ridges that initially form circumscribednot only the slightly tube (Figure2C) protruding whichlips of is the laterally slit but delimited also define by the sameshape glottal of the ridges pharyngeo-esophageal and lined by ciliated duct columnar lumen epitheliumcaudally, a Samples were rinsed in physiological saline and fixed in (Figure 2 B, C). The distance between the termination of the slit withphosphate hematoxylin buffered and 10% eosin formalin. (H&E) Tissuesand Alcian were blue processed stain. Immunohistochemistryand embedded in paraffin, for smooth sectioned muscle at actin 4 μm, was and performed stained esophageal duct, averaged 5mm in our specimens and was on sections using a Dako Autostainer using a biotinylated comprisedaperture and of thea tubularbeginning structure of the gas lined bladder, by epitheliumthe pharyngeo- and surrounded by a complete investment of soft tissue (duct). The chromogen and hematoxylin counterstain. A monoclonal mouse, duct continues caudally past the slit as a discrete tubular lumen, streptavidin antibody detection system (Dako LSAB2) with DAB terminating immediately as the lumen opens into a spacious

anti-human smooth muscle actin primary antibody was used of the slit are similar to the surrounding mucosa, being slightly (Dako Clone 1A4, code M0851, 1:100 dilution) and for a negative thickenedgas bladder and (Figure blending 1D, imperceptibly E, F, G). Grossly, into the them. surface The alligator margins control, mouse IgG1 was used. Deparaffinization was followed gar gas bladder lies caudal to the abrupt termination of the by hydration (1x Tris pH 6.0) for 5 minutes, then steamed for glottal ridges. 30 minutes (antigen retrieval), with subsequent automated processing with primary antibody, and DAB chromogen. Slides Citation: Page 2 of 8

Omar-Ali A, Baumgartner W, Allen PJ, Petrie-Hanson L (2016) Fine Structure of the Gas Bladder of Alligator Gar, Atractosteus Spatula. Int J Sci Res Environ Sci Toxicol 1(1): 8. Fine Structure of the Gas Bladder of Alligator Gar, Atractosteus Spatula Copyright: © 2016 Omar-Ali, et al.

for aGrossly, narrow themidline bladder’s strip of rich poorly vascular vascularized supply tissue can be running easily appreciated with finely arborescent vessels throughout, except continuous median central canal that divides the organ into along its ventral length (Figure 1A, B). This strip delineates a

symmetrical halves (Figure 1A, B, and F). The roof and the floor of this central canal are delicate, flat, fibrous membranes. The roof blends into the fibrous raphe that attaches the organ to the aortaEach (Figure half 1G).of the gas bladder is subdivided by smoothly

interconnected, progressively finer and shorter septa forming

Figure 2: Atractosteus spatula A

right, esophagus transected at the, pharyngeo-esophageal left, the ventral portion duct. of the: organ Gross removed.view of the Blue dorsum line ofindicates the pharyngeo-esophageal the level of sectioning junction, shown head in B. is Purple to the line indicates the level of sectioning shown in C. Green line indicates the B

level of sectioning shown in D. Formalin fixed specimen. : Photomicro- liumgraph, (black transverse arrow sectionand inset) of pharyngeo-esophageal connects the gut lumen slit (bottom) denoted to bythe blue be ginningline in figure of the A. duct A slit-like (top, green opening arrow). lined The by glottal a thick ridges transitional are denoted epithe by- the green, blue, and yellow arrows, which point to the epithelium, connective tissue stroma, and muscle bundles, respectively. Hematoxylin C -

and eosin, 20 x. : Photomicrograph, transverse section of pharyngeo- arrowesophageal and inset). duct denoted The lumen by ispurple surrounded line in figureon all sidesA. The by inner thick aspect myocol is lined by a tall simple to pseudostratified columnarD epithelium (black - lagenous stroma. Hematoxylin and eosin, 20 x. : Sub gross photomicrograph, transverse section of pharyngeo-esophageal duct denoted by terisks.green line A ringin figure of skeletal A, at themuscle duct and termination. tracts of smoothThe small muscle black delimit arrow theindicates duct. Thethe largetermination black arrow of the denotes glottal ridges,the esophagus, as seen inthe figure green 1 arrow D, as- denotes the air bladder proper, hematoxylin and eosin.

composedfirst, second, of third,dense and stroma fourth (Figure order alveolar3B). The chambers central canal(Figure is Figure 1: Atractosteus spatula. A laterally1C, E, G). delimited Septa tend by the to primary have abrupt septa, bulbousforming terminationsan arcade of ing organ (ABO), with gills, body wall, and other viscera removed. The regular pillars with wide intervening ostia that lead into the head is to the right. Arrow indicates: Ventro-dorsal the central view lumen, of theasterisks air-breath indi- gas exchange spaces. The progressing septal order divisions B view of the ABO, fresh specimen, and head is to the right. Arrow and- often form oblique angles, with a reduction in septal height asteriskscate the air denote exchange features fields. similar Formalin to that fixed in specimen.A. C : Ventro-dorsal the ABO, with the ventral half of the organ removed. Head is to the top of the page. Arrow denotes the central canal, dorsal: Ventro-dorsal aspect. Asterisks view of by approximately half, finally terminating in low alveoli D Microscopicapproximately Morphology 1 millimeter wide and (Figure Ultra structure1C, E).

denote primary and secondary septa. Formalin fixed specimen. : Cau- terminationdo-rostral view of the of theglottal ABO, ridges, transversely arrow indicates sectioned the approximately gas exchange 2cmsur In the glottis and pharyngeo-esophageal duct, the from the end of the pharyngeo-esophageal duct. Asterisks denoteE the - containspharyngeal large mucosa mucus is cellsstratified (Figure columnar 2B, C). epitheliumThe glottal thatridges varies are face. Dorsum is at the top of the page. Formalin fixed specimen. : Dor- thein thickness protruding from lips 100 of tomucosa 200 micrometers that guard theand entrance predominantly to the so-ventral view of the ventral half of the ABO, with the dorsum removed (opposite portion of the organ compared to C). Arrow denotes the floor specimen. F the duct forms a “T” where a 2 to 3 millimeter long stem dorsally lobeof the (arrow), central septa canal. (asterisks), Asterisks the denote central primary canal septa.is completely Formalin opened fixed meetspharyngeo-esophageal a collapsed tubule duct. forming Anteriorly the bar onof the transverse “T” (Figure section, 2B). : Caudo-rostral view of the ABO, transverselyG sectioned; left graph of a transverse section of the entire ABO, dorsum towards the Progressing caudally as the slit opening ends (Figure 2C), the top.in comparison Hematoxylin to and figure eosin. D. Formalin fixed specimen. : Photomicro- duct continues to meet the gas bladder proper.

Citation: Page 3 of 8

Anteriorly, pharyngeal mucosa lines the duct. Initially,

appears in the stem of the duct, becoming the predominant cell typea ciliated caudally stratified as the duct columnar becomes epithelium detached (esophageal from the ventral type) esophagus and eventually entirely lining the duct (Figure. 2B, C, D).

cushion of mucinous areolar connective tissue containing evenly In the alligator gar pharyngeo-esophageal duct, a thick

thisspread, stroma fine collagenblends into fibrils dense with plumpfascicles stellate of skeletal mesenchymal muscle admixedcells supports with smallthe duct nerves mucosa’s and vessels. (Figure. A thin2B and layer C). of Subjacently, transverse and obliquely arranged skeletal muscle bundles overlie the dorsum of the duct where it abuts the vertebral column, along with symmetrical, anteroposteriorly arranged thick muscle bundles (dorsal retractors).

ciliatedThe floorcolumnar of the epithelium central canal with and a prominent ventral aspects mucus of cell the primary septa are lined by a stratified to pseudo stratified, blue positive content, consistent with acid mucopolysaccharide (Figurecomponent. 3I). The mucus cells contain PAS (not shown) and Alcian

gradually thins, with eventual loss of ciliated cells, giving way to aProgressing simple squamous from first epithelium to fourth order covering septa, capillaries the epithelium and relatively few, evenly spaced, attenuated mucus cells (Figure 3C, D, E). This progression continues with eventual loss of mucus cells in the respiratory exchange surface (respiratory epithelium) (Figure 3E, F, G) (Figure 5A). This progressive epithelial change also occurs centrally to the periphery, where respiratory epithelium covers all alveolar surfaces; typical Figure 3: Multiple views of Atractosteus spatula. A pneumocytes occur here. The respiratory surface is covered by muscles, blood vessels, and forms bundles (arrow): Anthat overview are projecting of the towardleft lobe the shows lumen; the lumen lumen (L). and B septa. Septa are composed of striated micrographs demonstrate that squamous cells contain numerous irregular microvilli on squamous cells (Figure 4A, B, C). Electron levels of air spaces (asterisk) by series of septa (arrowhead); bundles of striated muscle are in the apical: portionEach lobe of septais subdivided (arrow). intoC and different D connections are composed of tight junctions and desmosomes withlamellar marked bodies invagination with stacked of content both adjacent (Figure 4C).cell Intercellularmembranes. lumnar epithelium with prominent mucus cell components (asterisk).: The Einternal surface is lined by a stratified to pseudostratified, ciliated co- lium rich with blood vessels (asterisk) (curved arrow).F Nuclei are irregular with a distinct thin rim of heterochromatin, blue: The stain internal section surface showing gradually the same changes G to simple squamous epithe- (Figure 5A). : resin toluidine abundant euchromatin, and prominent nucleoli (Figure 4A, B, C) rich with blood vessels with few, evenly spaced, attenuated mucus cells (arrow); resin toluidine blue stain section.:Simple H squamous epithelium Mucus cells lining alveoli and septal walls included occasional smooth muscle actin stain; smooth muscle (asterisk) is highlighted by Mucus cell content was hyper chromatic (Figure 4 A, B, C). the antigen, and interdigitates with striated :muscle Cross section (arrowhead); stained veins with are also lined by smooth muscle (double arrow). The internal surface is Neuroendocrine cells (NE) either between cells or along the with prominent mucus cells components (arrow). I: Mucus cells con basal aspects (Figure 4A). Neuroendocrine cytoplasm contained tainlined Alcian by a stratifiedblue positive to pseudostratified, content (thin arrow), ciliated striated columnar muscles epithelium (arrow many dense uniform granules approximately 100 nm wide. - Occasionally, NE cells were present in small tightly packed - groupsThe associated central canal, with septa, nerves and (Neuroepithelial alveoli consist Bodies-NEBs).of a similar sub head); nerve fiber (thick arrow); (Alcian Blue stain). epithelial stroma, composed of a thin layer of densely packed smooth muscle is present throughout the stroma at all levels andalong in their general, proximal forms aspects delicate (Figure fascicles 3H). and Unlike individualized skeletal muscle, cells of the septa, in particular the bulbous terminae, are composed of that admix with connective tissues. Muscle content gradually denselycollagen packed, fibers with irregularly fibroblasts arranged and small fascicles blood of vessels. striated The skeletal bulk decreases caudally and peripherally. septal skeletal muscle bundles with particular prominence muscle (Figure 3I). Smooth muscle is also prominent, investing Vessels, nerves, and unmyelinated axons run along the Citation: Page of 8

Omar-Ali A, Baumgartner W, Allen PJ, Petrie-Hanson L (2016) Fine Structure of the Gas Bladder of Alligator Gar, Atractosteus 4 Spatula. Int J Sci Res Environ Sci Toxicol 1(1): 8. Fine Structure of the Gas Bladder of Alligator Gar, Atractosteus Spatula Copyright: © 2016 Omar-Ali, et al.

apical portions of the septal terminae, typically subjacent to the

Capillaries are regularly nestled between epithelial cells and are coveredmuscle bundles apically (Figure. by thin 3A, extensions B, F, G, I) (Figure.of epithelial 4A, D) cells, (Figure. forming 5C).

B). The endothelium is continuous, overlying a collagenous lamina.an air-blood Endothelial barrier nucleiapproximately are located 1 micron away thickfrom (Figure.the surface 4A, and have irregular nuclei with rims of heterochromatin (Figure.

cavity, regular paired ganglia travel along the length of the organ4A, B). bilaterally. Within the Progressively delicate stroma smaller of thenerves floor without of the neurons central

small, random aggregates of lymphocytes that surround blood vesselsare present and encroach in the septa on the as epithelium they become with finer. occasional There areas are few of epithelial intercellular migration.

Figure 5: Alligator gar; transmission electron microscope (TEM) micro graphs of the ABO. A cleus adjacent to the border of the epithelium. The double arrow heads- indicates the basal cytoplasmic: The white extension arrows indicate just beneath the pneumocyte them. Filament nu- bundle of smooth muscles (white arrowhead) show sub plasmalem mal condensations (black arrow). B - cytoplasm, and the neuroendocrine :is This embraced TEM micrograph with an extension shows the of neuroendocrine cell with dense bodies/ granulesC (white arrow) in its rowhead) located between smooth muscles (black arrow) and striated musclesfibroblast (white (black arrow). arrowhead). The inset Lumen (TEM (L). micrograph : Unmyelinated under higher axons mag (ar-

broblast (asterisks). - nification) shows the unmyelinated axons that are embraced by the fi- Discussion

bladder, or ABO, is an unpaired oval elongated structure arising fromIn the air posterior breathing side fish, of thethe generalpharynx description and connected of the to gasthe Figure 4: Atractosteus spatula; Transmission electron micrographs of the gas bladder. A pharynx. It is located dorsal to the body lumen and consists of a consists of an outer microvillus border (thin black arrow) with a thin central canal that occupies a third of the gas bladder and two lobes cytoplasmic layer :(thin The capillarywhite arrow), barrier overlying in the respiratory a capillary epithelium(C) with a continuous endothelial lining (black arrow) and interstitial layer in be tween (asterisks).A red blood cell (RBC) is present in the lumen. The that are covered with a very thin tissue wall [1,3,22,24,25,37]. - Our findings in alligator gar were similar. The gas bladder was a large elongate organ with progressively finer and shorter septa thin white arrowhead indicates smooth muscle with filament bundles. heads and thick white arrow indicates nucleus) are present. B forming first, second, third, and fourth order alveolar chambers bladderA nerve fiberepithelium (N), and includes a neuroendocrine mucus cells cell (white (delimited arrowhead) by black and arrow epi- are also described to have trabeculae dividing the gas bladder thelial cells with numerous microvilli (thin black arrow); mucus: The nuclei gas and a central canal. The gas bladders of other air breathing fish (white arrow). L denotes the lumen. A Red Blood Cell (RBC) is in the- gas bladder is medially divided into two alveolate portions by capillary. Multi lamellar bodies (white asterisk) are located in the apical ainto central compartments canal lined and by a abundantcentral canal ciliated [1,30]. epithelium, The alligator similar gar

heads), and striated muscle (black arrow) are present within a loose part of the respiratory epithelium. Smooth muscle cells (black arrow- abundance of ciliated cells suggests that these tissues function collagenous stroma. C to bowfins and other gar species [24,29,38]. The location and multi lamellar bodies (thick arrows), lumen (L), dense bodies (black ar as a pulmonary escalator and move cell and foreign debris to the rowheads), microvilli (thin: The black apical arrows), region ofand the a pneumocyte epithelium includesnucleus duct and then out into the pharynx. (white arrowhead). D - is composed of loose collagenous stroma populated by smooth muscle (black arrow) and striated: (Overlapping muscle (white field arrow) with A) with The prominent lamina propria mito L. oculatusNumerous mucus cells and surfactant bodies were - identified in the respiratory epithelium, similar to that seen in chondria (arrowhead). Nerves (N) are separated from the other compo- [24]. The mucus content in mucus cells was an acid nents by fibroblast extensions (asterisk). mucopolysaccharide, similar to that seen in non-respiratory Citation: Page 5 of 8

airway surfaces in reptiles and mammals. Microvilli were abundant on the gas bladder surfaces of A. spatula, as was a secreted mucus barrier. We believe the functions of the the Inmucus alligator cells gar,and weciliated observed cells in that the Neuroendocrinerespiratory epithelial Cells microvilli, or ciliated cells, was to secrete mucus to protect the (NECs) are located between epithelial cells and also between internal surface of the gas bladder as has been suggested in other gar [39]. ofcells neuroendocrine as Neuroepithelial cells suggests Bodies NEBs.neuronal Similar control results of the were gas observed in spotted gar and long nose gar [24,38]. The presence Throughout the organ, smooth and skeletal muscle fascicles bladder [37,38]. The internal surface of the alligator gar gas bladder is rich organ. The muscle fascicles were often intimately associated in ciliated cells that are irregular in shape and project toward were abundant, giving strength to this delicate, well-vascularized with one another. The intermediate layer of the Lepisosteus gas the lumen of the gas bladder. These ciliated cells reduce the area bladder was described to consist of striated muscle, smooth ciliated cells has been observed in spotted gar and amphibians bundles in the connective tissue of the alligator gar gas bladder. available for respiratory exchange. Similar distribution of these muscle, fibroblasts, and elastic fibers [22]. We observed nerve [24,48,50].Alligator gar neuroendocrine cells were similar to those These findings were also reported in the spotted gar gas bladder [24]. Nerve fibers were also found in the walls of the ABO of bichirThe and nature gars [40,of the 41]. opening to the gas bladder is debated in theobserved respiratory in the epithelium. ABO of otherThese gars.cells were Neuroendocrine characterized Cells by the(NECs) presence were of pyramidal dense secretory and occurred granules, in theGolgi basal bodies, portion rough of Lepisosteus endoplasmic reticulum, and scattered numbers of mitochondria ofgars a pneumatic [24,29]. It hasduct beenin Atractosteus anatomically tristoechus described in multiple species [1,24,29,38]. There is one brief description work in L. oculatus and L. osseus conclude that, in Lepisosteus, cells in the gas bladder indicates endocrine and paracrine [42]. Recent in the basal part of the cell [46]. The presence of neuroendocrine like opening and that a discrete tubular structure, or “duct” is the alimentary canal opens into the gas bladder through a slit- control over bladder function [37,38,46]. Neuroepithelial bodies and NEs are surrounded and invested by ciliated cells, goblet lacking [24,29]. In our study, we found that alligator gar have cells,The and alligator pneumocytes gar air [37,38,46]. breathing organ is a single air sac that epitheliuma pharyngeo-esophageal lined lumen within duct. a tubular It is anteriorly structure delimited circumscribed by a emerges from the dorsal margin of the foregut, while amphibian byslit-like connective opening tissue. and This a slender location aperture is similar that to continuesthe gas bladder as an lungs are paired organs that emerge from the ventral margin of opening described in L.oculatus L. osseus, in which the gas bladder gradually widens from a narrow anterior point, the gas bladder in A. spatula begins, [24]. where Unlike an abrupt termination alligatorthe foregut gar [25,48]. ABO has There a series are of many air spaces similarities similar in to the the anatomy lung but and histology of the gar ABO and amphibian lungs [24].The a fully dilated, alveolated gas bladder with squamous respiratory structure of the alligator ABO is very similar to the ABO of the epithelium.of the glottal This ridges epithelium of the pharyngeo-esophageal is continuous with the duct central opens canal into genusit lacks Lepisosteus the bronchial. Alligator tree that gar is inhabitsobserved coastal in lungs estuaries [51,52]. thatThe of the gas bladder. The glottal ridges of A. spatula are similar to are prone to agricultural and petrochemical pollution. One study those described in L. oculatus and L. osseus reported that Atractosteus tropicus and Lepisosteus oculatus had of robust paired dorsal retractor muscles is also similar, and may control the size of the glottal opening. [24,29]. The presence had low, acceptable levels of contaminants [53]. Therefore, these higher tissue PCB concentrations than other fish in waters that In Lepisosteus, the interior surface of the respiratory epithelium is ciliated and lined with lamellar bodies and goblet pollutants on aquatic ecosystems. fish are an excellent sentinel model for studying the effects of cells [22]. In mammals, respiratory alveolar surfaces are lined by References type I and type II pneumocytes. Type I cells are simple squamous

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Omar-Ali A, Baumgartner W, Allen PJ, Petrie-Hanson L (2016) Fine Structure of the Gas Bladder of Alligator Gar, Atractosteus Spatula. Int J Sci Res Environ Sci Toxicol 1(1): 8.